Method for detection of fibronectin receptor ligands

ABSTRACT

The present invention provides a substantially pure integrin characterized in that it consists of an  alpha v and a  beta 1 subunit. The receptor binds to fibronectin and GRGDSPK but does not bind to vitronectin. The  alpha v beta 1 receptor can be used to determine the presence of ligands for the receptor.

This work was supported by grants CA 42507-03 and CA 28896-08 and Cancer Center Support Grant CA 30199-08 from the National Cancer Institute. The United States Government has certain rights in this invention.

This application is a continuation of application Ser. No. 07/942,582, filed Sep. 9, 1992, now abandoned, which is a divisional of application Ser. No. 07/461,349, filed Jan. 5, 1990, U.S. Pat. No. 5,169,930.

BACKGROUND OF THE INVENTION

This invention relates to receptors for adhesion peptides, and more specifically to a novel receptor having affinity for fibronectin.

Multicellular organisms, such as man, have some 10¹⁴ cells which can be divided into a minimum of fifty different types, such as blood cells and nerve cells. During the course of growth and development, cells adhere to other cells, or to extracellular materials, in specific and orderly ways. Such cell adhesion mechanisms appear to be of importance in mediating patterns of cellular growth, migration and differentiation, whereby cells develop specialized characteristics so as to function as, for example, muscle cells or liver cells. Cell adhesion mechanisms are also implicated in dedifferentiation and invasion, notably where cells lose their specialized forms and become metastasizing cancer cells.

The mechanisms underlying the interactions of cells with one another and with extracellular matrices are not fully understood, but it is thought that they are mediated by cell surface receptors which specifically recognize and bind to a cognate ligand on the surface of cells or in the extracellular matrix.

The adhesion of cells to extracellular matrices and their migration on the matrices is mediated in many cases by the binding of a cell surface receptor to an Arg-Gly-Asp containing sequence in the matrix protein (as reviewed in Ruoslahti and Pierschbacher, Science 238:491(1987)). The Arg-Gly-Asp sequence is a cell attachment site at least in fibronectin, vitronectin, various collagens, laminin and tenascin. Despite the similarity of their cell attachment sites, these proteins can be recognized individually by the specific receptors.

Integrins are a family of adhesion receptors which bind to Arg-Gly-Asp binding sites of extracellular matrix membrane proteins via the Arg-Gly-Asp binding sites. They are heterodimeric molecules composed of one alpha (α) and one beta (β) subunit. Several subunits of each kind are known, and various αβ combinations make up receptors with different ligand specificities.

Eleven distinct alpha chains have thus far been described. Formerly, they have been divided into three main subfamilies based on the beta subunit with which they associate. The β₁ subfamily includes receptors for fibronectin, various collagens, laminin and tenascin. The β₂ subfamily consists of leukocyte specific receptors, while the β₃ subfamily contains multispecific receptors commonly referred to as the platelet glycoprotein IIb-IIIa and the vitronectin receptor. Among the combinations known to exist, the α_(v) subunit associates with the β₃ subunit to form a vitronectin receptor and with two recently described β subunits called β_(x) and β_(s). The α_(v) β_(x) integrin is a vitronectin and fibronectin receptor while the ligand specificity of α_(v) β_(s) is not known.

Because of the importance of integrins in mediating critical aspects of both normal and abnormal cell processes, there exists the need to identify and characterize different integrins. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides a substantially pure integrin-type receptor characterized in that it consists of an α_(v) and a β_(l) subunit. The α_(v) β₁ integrin binds to fibronectin and GRGDSPK but does not bind to vitronectin. The α_(v) β₁ integrin can be used to determine the presence of a α_(v) β₁ ligand and to develop adhesion peptides specific for the various integrins. The presence of the α_(v) β₁ can be used to assess ability of cells to adhere to fibronectin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of a gel showing integrin subunits expressed on various cell types.

FIGS. 2A and 2B show the results of cell adhesion assays on fibronectin and vitronectin. The error bars indicate the standard error of the mean of three independent assays. Figure legend: -▪- designates IMR 32 cells and -- designates MG-63 cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a new receptor composed of an α_(v) and a β₁ subunit, or their immunological equivalents. This integrin-type receptor is herein termed "α_(v) β₁ receptor" or "α_(v) β₁ integrin." The α_(v) β₁ receptor is immunoprecipitated with a monoclonal antibody to the α_(v) subunit and includes a band in the expected position of the β₁ subunit, as shown in FIG. 1, left panel.

To confirm the association between the α_(v) and β₁ subunits implied by the immunoprecipitation results described above, monoclonal antibodies to each of the subunits were used to isolate receptor complexes from the fibroblast cell line WI-38. A series of antibodies were then used to identify the co-isolated subunit. The material purified by the anti-α_(v) monoclonal antibody was precipitated by two different anti-β₁ monoclonal antibodies and by a polyclonal serum to a peptide representing the β₁ cytoplasmic domain. All three anti-β₁ reagents recognized the α_(v) -containing integrin. Conversely, the material obtained with a β₁ monoclonal antibody was immunoprecipitated by two different anti-α_(v) monoclonal antibodies and by a polyclonal serum to a peptide representing the α_(v) cytoplasmic domain. These data indicate that the α_(v) and β₁ subunits do associate to form a complex.

To investigate the ligand binding specificity of the new α_(v) β₁ integrin, affinity chromatography experiments and cell adhesion assays were performed. In the chromatography experiments, detergent extracts of IMR 32 neuroblastoma cells surface labelled with ¹²⁵ I were fractionated on a 110 kD fibronectin fragment and GRGDSPK peptide affinity columns. The α_(v) β₁ integrin bound to a 110 Kd fragment of fibronectin that contains the cell attachment site. It was eluted from the column with a peptide (GRGDSP), that represents the cell attachment site, but not a related peptide, GRGESP. No additional bands appeared with subsequent EDTA elution. The receptor also bound to a column containing the peptide GRGDSPK coupled to Sepharose and was eluted with the GRGDSP peptide but not with the GRGESP peptide.

Amino acids are identified herein by the standard one-letter abbreviations, as follows:

    ______________________________________                                         Amino Acid     Symbol                                                          ______________________________________                                         Alanine        A                                                               Aspartic acid  D                                                               Cysteine       C                                                               Glutamine      Q                                                               Glutamic acid  E                                                               Glycine        G                                                               Histidine      H                                                               Isoleucine     I                                                               Leucine        L                                                               Lysine         K                                                               Methionine     M                                                               Phenylalanine  F                                                               Proline        P                                                               Serine         S                                                               Threonine      T                                                               Tryptophan     W                                                               Tyrosine       Y                                                               Valine         V                                                               ______________________________________                                    

To confirm that the eluted material was the α_(v) β₁ complex, the peak fractions from each column were pooled and immunoprecipitated with monoclonal antibodies to the α_(v) and β₁ subunits. Both antibodies precipitated the same two bands from each column, indicating that the material specifically eluted from each column was, in fact, α_(v) β₁.

Cell attachment assays showed that the IMR 32 cells attached to fibronectin but not to vitronectin (FIG. 2A) or fibrinogen (data not shown). The adhesion of these cells to fibronectin appears to be mediated by the α_(v) β₁ complex since the only other integrin detected, α₁ β₁, did not bind fibronectin in the affinity chromatography experiments (see FIG. 1). These cells also attached to collagens I and IV and laminin presumably due to the presence of the α₁ β₁ complex.

The data indicate that the α_(v) β₁ integrin subunits associate to form a functional fibronectin receptor. Although molecular heterogeneity due to modifications such as alternative splicing cannot be entirely ruled out, the subunits of the new receptor were immunologically indistinguishable from α_(v) and β₁ with at least three antibodies to each subunit. Therefore, by the criteria of electrophoretic mobility and immunological reactivity, the new receptor is composed of α_(v) and β₁ subunits or their immunological equivalents.

The new α_(v) β₁ does not bind to vitronectin but can be isolated on a GRGDSPK column. This ligand binding pattern appears to be different from that of any of the previously characterized integrins. The ability of this receptor to bind to a GRGDSPK column is a property shared by two vitronectin-binding integrins, α_(v) β₃ (Pytela, et al., Proc. Natl. Acad. Sci. USA 82:5766 (1985)), and the platelet receptor α_(IIB) β₃ (Pytela, et al., Science 231:1559 (1986) (and references therein), which are incorporated herein by reference). A complex between α_(v) and the recently described β_(s) subunit may also belong to this group (Freed, et al., EMBO 8:2955 (1989), which is incorporated herein by reference). Another recently described complex of α_(v) (α_(v) β_(x) ) binds to both fibronectin and vitronectin (Cheresh et al., Cell 57:59 (1989), which is incorporated herein by reference).

Fibronectin-binding integrins of the β₁ class (α₅ β₁) do not bind to vitronectin and unlike the α_(v) β₁ integrin described here, do not bind detectably to the GRGDSPK column. Therefore, the α_(v) β₁ complex appears to have a distinct intermediate specificity between the vitronectin binding integrins and the β₁ class integrins.

Three different α subunits have been shown to associate with more than one β subunit. Two of these, α₄ and α₆, can pair with either one of two β subunits. The α_(v) subunit appears to be especially versatile since it has already been shown to be capable of associating with four β subunits. Moreover, the association between α_(v) and β₁ described here unexpectedly crosses the boundaries of two previously proposed integrin classes, forcing a reevaluation of the currently accepted integrin classification.

Since receptors for collagens, laminin and fibronectin all share a common β subunit, it has been proposed that the α subunit determines the specificity of integrins. The new α_(v) β₁ integrin described here is a fibronectin receptor, whereas α_(v) β₃ is a vitronectin receptor. This result, along with the demonstration that α_(v) β_(x) binds to fibronectin, shows that the β subunit plays a greater role in determining receptor specificity than thought previously.

The α_(v) β₁ is useful in assaying the ability of cells to attach to extracellular matrices; the presence of α_(v) β₁ on the cell surface indicates the ability to attach to fibronectin. The presence of the α_(v) β₁ integrin is detected in an immunoassay format using an antibody against each of the subunits as described in Example I or by a modification of that immunoassay format. Such assays are well known to those skilled in the art. See generally, ANTIBODIES; A LABORATORY MANUAL (Harlow and Lane, eds.) Cold Spring Harbor Laboratory (1988), which is incorporated herein by reference.

Another area where the α_(v) β₁ receptor is useful is the analysis of ligands for integrins. The specificity of such ligands is important. For example, synthetic peptides containing the RGD sequence that bind to the platelet integrin gp IIb/IIIa but not to other integrins are being developed into anti-platelet pharmaceuticals.

The ability of a compound to interact with the α_(v) β₁ integrin can be assessed by affinity chromatography as described under Example II. A cell attachment assay can be used as described under Example III when the contribution by other integrins possessed by the test cells can be excluded. Finally an enzyme immunoassay format or a radioreceptor assay can be used as described in Hautanen et al., J. Biol. Chem. 264:1347-1442 (1989).

The following examples are intended to illustrate but not limit the invention.

EXAMPLE I Identification of a_(v) β₁ Integrin

Antibodies to the integrin subunits were prepared as indicated in the following table:

                  TABLE I                                                          ______________________________________                                                      mono-                                                                          clonal              reference                                     sub-         or poly-            or                                            unit host    clonal   immunogen  confirmation                                  ______________________________________                                         α.sub.v                                                                       mouse   mono-    purified   immunoblotting;                                            clonal   vitronectin                                                                               reactive with                                              Mab 147  receptor   α.sub.v subunit                         α.sub.v                                                                       mouse   Mab 59   purified   immunoblotting;                                                     vitronectin                                                                               reactive with                                                       receptor   α.sub.v subunit                         α.sub.v                                                                       rabbit  poly-    KRVRPPQEE- Freed et al.                                               clonal   QEREQLQPH- EMBO J.                                                             ENGEGNSET  8:2955 (1989)                                 α.sub.5                                                                       rabbit  poly-    EKAQLKP-   immunoblotting;                                            clonal   PATSDA     reactive with                                                                  α.sub.5 subunit                         α.sub.6                                                                       mouse   mono-    α.sub.6                                                                             Sonneberg et al.,                                          clonal              J. Biol. Chem.                                             GoH3                263:14030 (1988)                              α.sub.2                                                                       mouse   mono-    α.sub.2                                                                             Wagner and Carter                                          clonal              J. Cell Biol.                                              PIH5                105:1073 (1987)                               α.sub.3                                                                       rabbit  poly-    cytoplasmic                                                                               Hynes et al.,                                              clonal   domain of  J. Cell Biol.                                                       α.sub.3 subunit                                                                     109:409 (1989)                                β.sub.1                                                                        rabbit  poly-    KKKEKEKMN- immunoblotting;                                            clonal   AKWDTGENP- reactive with                                                       IYSAVTTVV- β.sub.1 subunit                                                NPKYEGK                                                  β.sub.1                                                                        mouse   mono-    purified   immunoblotting;                                            clonal   fibronectin                                                                               reactive with                                              LM 534   receptor   β.sub.1 subunit                                       LM 442                                                            ______________________________________                                    

Human neuroblastoma cells (IMR 32; ATCC Accession No. CCL 127), lung cell fibroblasts (WI-38; ATCC Accession No. CCL 75), for example, (WI-38; ATCC Accession No. CCL 757) and glioblastoma cells (U251) were surface labeled with ¹²⁵ I and lactoperoxidase according to the method of Pytela et al., Cell 40:191-198 (1985), which is incorporated herein by reference, and extracted with a buffer containing a 0.5% Triton-X-100, 150 mM NaCl, 1 μg/ml leupeptin, 1 mg/ml aprotinin, 0.4 μg/ml pepstatin and 10 mM Tris, pH 7.2. Integrin heterodimers were immunoprecipitated with antibodies specific for either the β₁ or α subunits and analyzed by SDS-PAGE. Briefly, the extracts were clarified at 15,000 rpm and precleared by an incubation with preimmune rabbit or mouse IgG-Sepharose. Following an incubation with the primary antibodies, immunocomplexes were uncovered with either Sepharose-Protein A or Sepharose-goat anti-mouse IgG.

α_(v) -containing integrins and β₁ -containing integrins were immunopurified from the WI-38 extract on anti-α_(v) (Mab 147) and anti-β₁ (Mab LM 534) Sepharose columns respectively. The column was eluted with 50 mM glycine-HCl pH 3, containing 0.5% Triton-X-100. After neutralization, the material was divided in three aliquots for immunoprecipitation with anti-β₁ antibodies or anti-α_(v) antibodies and the immunoprecipitates were analyzed by SDS-PAGE substantially as described above. In each case association between the α_(v) and the β₁ subunits was found.

EXAMPLE II Analysis of Ligand Specificity and Purification of α_(v) β₁ Integrins

IMR 32 cells were surface labeled with ¹²⁵ I and lysed i1n 200 mM octylglucoside, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 0.4 μg/ml pepstatin, and 10 mM Tris, pH 7.2. The cell extract was applied to a 110 kD fibronectin fragment-Sepharose column and the column was washed with 50 mM octylglucoside, 1 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂, 150 mM NaCl, and 10 mM Tris, pH 7.2, alone and with 1 mg/ml GRGESP peptide. The column was subsequently eluted with 1 mg/ml GRGDSP peptide followed by elution with 10 mM EDTA. IMR 32 cell extract was also fractionated by identical means on a GRGDSPK column.

These procedures were similar to those described in Pytela et al., Meth. Enzymol. 144:475-489 (1987); and Gailit and Ruoslahti, J. Biol. Chem. 263:12927-12932 (1988), which are incorporated herein by reference. The eluates from each of these columns contained an integrin with an α and a β subunit. The peak fractions were pooled and immunoprecipitated with the anti-α_(v) (Mab 147), or anti-β₁ (Mab LM 534) described in Example I. The integrin bound to the column was found to precipitate with both anti-α_(v) and anti-β₁, indicating that it is an association of the α_(v) and β₁ subunits.

The Gailit and Ruoslahti, J. Biol. Chem. 263:12927-12932 (1988) incorporated material is: Fibronectin receptor was purified from human placenta by affinity chromatography on a 110 kDa¹ chymotryptic fragment of fibronectin. The preparation of the affinity matrix and the isolation of receptor have been described in detail (Pytela et al., Cell 40:191-198 (1985), Pytela et al., Methods Enzym. 144:475-489 (1987)). One change from the original procedure was the use of Tris-buffered saline (TBS,² 150 mM NaCl, 50 mM Tris-HCl, pH 7.3), instead of a phosphate buffer, to ensure solubility of the divalent cations. Briefly, homogenized placental tissue was washed once with 2 volumes of cold TBS containing 1 mM CaCl₂, 1 mM MgCl₂, and 3 mM PMSF, centrifuged, and the pellet was extracted with 1 volume of cold TBS containing 100 mM octyl-β-D-glucopyranoside (Calbiochem, La Jolla, Calif.), and calcium, magnesium, and PMSF as above. The clear extract was slowly applied at 4° C., first to a column of plain Sepharose, and then to 110-kDa fibronectin fragment-Sepharose. The affinity column was washed at room temperature with 6 volumes of TBS containing 25 mM octyl-β-D-thioglucopyranoside (Calbiochem), and 1 mM CaCl₂ and MgCl₂. Bound receptor was eluted with the wash buffer containing 20 mM EDTA in place of calcium and magnesium. Eluted receptor was used immediately for the preparation of receptor liposomes or stored frozen. In some experiments, 1 mM MnCl₂ was substituted for CaCl₂ and MgCl₂ throughout the isolation procedure.

The Pytela et al., Meth. Enzymol. 144:475-489 (1987) incorporated material is: An affinity matrix is prepared by coupling to cyanogen bromide-activated Sepharose a 120-kDa chymotryptic fragment of fibronectin that binds to neither gelatin nor heparin but retains cell attachment-promoting activity (Pierschbacher et al., Cell 26:259 (1981), Engvall et al., Cell 29:475 (1982)). To prepare the fragment, fibronectin is isolated by gelatin-Sepharose chromatography from human plasma or plasma cryoprecipitate. (The infection risks should be noted in working with human plasma and plasma products.) The eluates from gelatin-Sepharose are dialyzed against PBS and digested with 1% chymotrypsin (w/w) for 1 hr at 37°. The digestion is stopped by adding phenylmethylsulfonyl fluoride (PMSF) to 10⁻⁴ M. The gelatin-binding and heparin-binding fragments are then removed by gelatin-Sepharose and heparin-Sepharose and the nonbinding fragments are fractionated on Sephacryl S-200 to remove any small fragments.

EXAMPLE III Cell Adhesion Assays

Microtiter plates were coated with various concentrations of fibronectin and vitronectin and postcoated with 0.05% bovine serum albumin. After washing, approximately 10⁵ IMR32 (human neuroblastoma; ATCC CCl 127) or MG-63 (human osteosarcoma; ATCC CCL 1427) cells were plated per well and incubated at 37° C. for 90 minutes. The attached cells were fixed in 3% paraformaldehyde and stained with 0.5% crystal violet. The attachment was quantitated by reading the absorbance at 600 nm. As shown in FIG. 2A, the IMR 32 cells attach to fibronectin, but not to vitronectin or fibrinogen (data not shown), whereas the MG-63 cells attach to the two substrates.

Although the invention has been described with reference to the presently-preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. 

We claim:
 1. A method useful for determining whether a compound is a ligand for α_(v) β₁ integrin comprising the steps of:(a) contacting the compound with substantially purified α_(v) β₁ integrin and (b) determining whether the compound binds to α_(v) β₁ integrin, the presence of binding indicating that the compound is a ligand for α_(v) β₁ integrin.
 2. A method useful for determining whether a compound is a ligand for α_(v) β₁ integrin comprising the steps of:(a) creating a sample mixture by contacting the compound with substantially purified α_(v) β₁ integrin under conditions that allow binding of α_(v) β₁ integrin to a ligand; (b) removing unbound α_(v) β₁ integrin from the sample mixture to produce a bound fraction of the sample mixture; and (c) determining whether α_(v) β₁ integrin bound to the compound in the sample mixture, the binding of which indicates that the compound is a ligand for α_(v) β₁ integrin.
 3. The method of claim 2 wherein the sample mixture comprises the compound attached to a solid support and step (c) comprises detecting the α_(v) β₁ integrin in the bound fraction, wherein detection of α_(v) β₁ integrin in the bound fraction indicates that the compound is a ligand for α_(v) β₁ integrin.
 4. The method of claim 3 wherein detecting α_(v) β₁ integrin in the bound fraction comprises immunoreacting the bound fraction with antibody.
 5. The method of claim 4 wherein the α_(v) β₁ integrin is detected by immunoreaction with anti-α_(v) and anti-β₁ antibody.
 6. The method of claim 5 wherein the α_(v) β₁ integrin is detected by sequential immunoreaction with anti-α_(v) and anti-β₁ antibody in either sequence. 